Download Kinesin-1–syntaphilin coupling mediates activity

JCB: Article
Published July 15, 2013
Kinesin-1–syntaphilin coupling mediates
activity-dependent regulation of axonal
mitochondrial transport
Yanmin Chen and Zu-Hang Sheng
A
xonal mitochondria are recruited to synaptic terminals in response to neuronal activity, but the
mechanisms underlying activity-dependent regulation of mitochondrial transport are largely unknown. In
this paper, using genetic mouse model combined with live
imaging, we demonstrate that syntaphilin (SNPH) mediates the activity-dependent immobilization of axonal
mitochondria through binding to KIF5. In vitro analysis
showed that the KIF5–SNPH coupling inhibited the motor
adenosine triphosphatase. Neuronal activity further recruited SNPH to axonal mitochondria. This motor-docking
interplay was induced by Ca2+ and synaptic activity and
was necessary to establish an appropriate balance between motile and stationary axonal mitochondria. Deleting snph abolished the activity-dependent immobilization
of axonal mitochondria. We propose an “Engine-Switch
and Brake” model, in which SNPH acts both as an engine off switch by sensing mitochondrial Rho guanosine
triphosphatase-Ca2+ and as a brake by anchoring mitochondria to the microtubule track. Altogether, our study
provides new mechanistic insight into the molecular interplay between motor and docking proteins, which arrests
axonal mitochondrial transport in response to changes in
neuronal activity.
Introduction
Neurons require specialized mechanisms to transport mitochondria to axons and to maintain their retention near synaptic
terminals, where energy production and calcium homeostatic
capacity are in high demand. The loss of mitochondria from
axon terminals results in impaired synaptic transmission (Guo
et al., 2005; Verstreken et al., 2005; Ma et al., 2009). Axonal
mitochondria display complex motility patterns characterized
by frequent pauses, changes in direction, and stationary docking (Hollenbeck and Saxton, 2005), suggesting that mitochondria are coupled to molecular motors kinesin-1 for anterograde
transport and dyneins for retrograde movement together with
docking machinery (MacAskill and Kittler, 2010; Sheng and
Cai, 2012). Kinesin-1 is a tetramer consisting of a homodimer
of one of three kinesin-1 heavy chains (KHCs; KIF5A, -B, and
-C) and two kinesin light chains (KLCs; KLC1 and KLC2;
Hirokawa et al., 2010). KIF5 motors use adaptors for cargo
recognition and binding. In Drosophila melanogaster, mitochondrial Rho GTPase (Miro; dMiro) connects mitochondria
to kinesin motors through Milton (Stowers et al., 2002; Guo
et al., 2005). In mammals, two isoforms of Miro (Miro1 and 2)
act similarly (Fransson et al., 2006). Trak1 and 2 (trafficking
kinesin-binding protein 1 and 2), mammalian Milton ortholo­
gues, serve as an adaptor linking mitochondria to KIF5 through
Miro1 (Smith et al., 2006; MacAskill et al., 2009a). Motile
mitochondria are recruited to synapses in response to elevated
Ca2+ and synaptic activity (Rintoul et al., 2003; Yi et al., 2004;
MacAskill et al., 2009b; Wang and Schwarz, 2009). Synaptic
structure and function are highly plastic and undergo activitydependent remodeling, thereby dynamically altering mitochondrial motility and distribution.
How are motile mitochondria recruited to the stationary
pool in response to synaptic activity? Recent work characterized Miro’s role as a Ca2+ sensor to regulate mitochondrial
Downloaded from on June 12, 2017
THE JOURNAL OF CELL BIOLOGY
Synaptic Function Section, The Porter Neuroscience Research Center, National Institute of Neurological Disorders and Stroke, National Institutes of Health,
Bethesda, MD 20892
Correspondence to Zu-Hang Sheng: [email protected]
Abbreviations used in this paper: DHC, dynein heavy chain; DIV, day in vitro;
ES, embryonic stem; KBD, KHC binding domain; KHC, kinesin-1 heavy chain;
KLC, kinesin light chain; Miro, mitochondrial Rho GTPase; Mito, mitochondria
targeting sequence; MT, microtubule; N-MD, N-terminal motor domain; PGK,
phosphoglycerate kinase; SNPH, syntaphilin; TTX, tetrodotoxin.
This article is distributed under the terms of an Attribution–Noncommercial–Share Alike–No
Mirror Sites license for the first six months after the publication date (see http://www.rupress
.org/terms). After six months it is available under a Creative Commons License (Attribution–
Noncommercial–Share Alike 3.0 Unported license, as described at http://creativecommons
.org/licenses/by-nc-sa/3.0/).
Supplemental Material can be found at:
/content/suppl/2013/07/12/jcb.201302040.DC1.html
The Rockefeller University Press $30.00
J. Cell Biol. Vol. 202 No. 2 351–364
www.jcb.org/cgi/doi/10.1083/jcb.201302040
JCB
351
Published July 15, 2013
Figure 1. SNPH is required for activity-dependent immobilization of axonal mitochondria. (A–D) Kymographs
(A and B) and quantitative analysis (C and D) showing
the motility of axonal mitochondria and late endosomes
along the same axons of snph+/+ and snph/ neurons
before and after treatment (2 min) with 50 mM KCl and
10 µM FPL 64176. Hippocampal neurons were cotransfected with DsRed-Mito and YFP-Rab7 at DIV9 followed
by imaging at DIV12. Vertical lines in kymographs represent stationary mitochondria; slanted lines or curves to
the right (negative slope) represent retrograde movement;
curves to the left (positive slope) indicate anterograde
movement (also see Fig. S1 and Videos 1, 2, and 3).
(E) The relative motility of dendritic mitochondria in
snph+/+ and snph/ hippocampal neurons before and
after treatment (2 min) with 50 mM KCl and 10 µM FPL
64176. Data were quantified from numbers of neurons
indicated in parentheses (C and D) or within bars (E) in
three independent experiments. Bars, 10 µm. Error bars
show SEM. Student’s t test.
Downloaded from on June 12, 2017
motility (Saotome et al., 2008; MacAskill et al., 2009b; Cai and
Sheng, 2009; Wang and Schwarz, 2009). Miro has two EF-hand
Ca2+ binding domains (Fransson et al., 2006). Activity-triggered
Ca2+ influx recruits motile mitochondria to the stationary phase
adjacent to activated synapses through a Miro-Ca2+ sensor that
inactivates KIF5-driven transport machineries. Although this
352
JCB • VOLUME 202 • NUMBER 2 • 2013
model is attractive, the mechanistic insight into how Miro-Ca2+
sensing inactivates KIF5 motor is unclear. Recently, we identified syntaphilin (SNPH) as a “static anchor” specific for axonal
mitochondria (Kang et al., 2008; Chen et al., 2009). SNPH
targets to axonal mitochondria and mediates their docking by
anchoring them to the microtubule (MT)-based cytoskeleton.
Published July 15, 2013
Downloaded from on June 12, 2017
Figure 2. SNPH is required to arrest axonal mitochondrial movement in response to synaptic activity. (A and B) Kymographs (A) and quantitative
analysis (B) showing the motility of axonal mitochondria in snph+/+ and snph/ hippocampal neurons before and after 100-Hz field stimulation in the
presence or absence of 1.2 mM CaCl2 or 1 µm TTX. Neurons were transfected with DsRed-Mito at DIV9 followed by time-lapse imaging at DIV12.
Vertical lines in kymographs represent stationary mitochondria; slanted lines or curves to the right (negative slope) represent retrograde movement (also
see Videos 4 and 5). Data were quantified from numbers of neurons indicated within bars in three independent experiments. Bars, 10 µm. Error bars
show SEM. Student’s t test.
Deleting snph in mice robustly increases axonal mitochondrial
motility. Thus, identifying SNPH as a docking protein provides
a molecular target to investigate how motile axonal mitochondria are recruited to the stationary pool in response to changes
in neuronal activity.
Using snph mouse models and time-lapse imaging analysis in live neurons, we demonstrate that SNPH mediates the
activity-dependent immobilization of axonal mitochondria by
physical displacement of KIF5 from the Miro–Track (trafficking kinesin-binding protein) complex. Such a KIF5–SNPH coupling inhibits KIF5 ATPase and is controlled by a Miro-Ca2+
sensing switch in response to neuronal activity. We propose the
“Engine-Switch and Brake” model, which nicely reconciles the
current dispute in explaining how Miro-Ca2+ sensing arrests
mitochondrial transport. Our study elucidates a new molecular
mechanism underlying the complex regulation of axonal mitochondrial transport, thereby advancing our knowledge that may
be essential for maintaining axonal and synaptic homeostasis.
Results
SNPH is required for activity-dependent
regulation of mitochondrial transport
To determine whether SNPH is involved in Ca2+-dependent immobilization of axonal mitochondria, we conducted time-lapse
imaging in live hippocampal neurons to record the transport of
both mitochondria and late endosomes along the same axons
of snph+/+ and snph/ neurons expressing DsRed–mitochondria
targeting sequence (Mito) and YFP-Rab7. To elevate cytosolic
Ca2+, the neurons were treated briefly (2 min) with 50 mM KCl
and 10 µM FPL 64176, an L-type Ca2+ channel agonist. This
combined application was shown to raise synaptic Ca2+ to levels similar to those produced by 30 µM glutamate and result in
an inhibitory effect on mitochondrial motility without inducing
neuronal toxicity (MacAskill et al., 2009b). As we previously
reported (Kang et al., 2008; Chen et al., 2009; Cai et al., 2010),
axons were distinguished from dendrites based on known morphological characteristics: greater length, thin and uniform
diameter, and sparse branching (Banker and Cowan, 1979). Kymographs were used to quantify stationary and motile organelles
(Miller and Sheetz, 2004). The KCl treatment in snph+/+ neurons decreased axonal mitochondria motility (35 ± 2% before
and 15 ± 2% after treatment; mean ± SEM, P < 0.001; Fig. 1,
A and C). In contrast, the same treatment of snph/ neurons
failed to affect axonal mitochondria motility (81 ± 5% before
and 76 ± 4% after treatment, respectively; P = 0.22; Fig. 1,
B and C). As a control, the KCl treatment had no significant
effect on the motility of late endosomes labeled with YFP-Rab7
(Fig. 1, A, B, and D; and Videos 1 and 2) or early endosomes labeled with GFP-Rab5 (Fig. S1, A and B) along the same axons
of snph+/+ and snph/ neurons. Furthermore, overexpressing
SNPH in wild-type neurons immobilized axonal mitochondria
but had no effect on late endosomes (Fig. S1 C and Video 3). In
dendrites, however, mitochondrial motility in both snph+/+ and
Regulation of axonal mitochondrial transport • Chen and Sheng
353
Published July 15, 2013
Figure 3. SNPH dominantly immobilizes axonal mitochondria. (A and B) Kymographs (A)
and quantitative analysis (B) showing axonal
mitochondrial motility in snph+/+ neurons expressing SNPH, Miro1, or Trak2 alone or coexpressing Miro1 + SNPH or Trak2 + SNPH.
Hippocampal neurons were transfected at
DIV9 and imaged at DIV12. Data were quantified from numbers of neurons (indicated in parentheses) in three independent experiments.
Bars, 10 µm. Error bars show SEM. Student’s
t test. Also see Fig. S2 and Videos 6 and 7.
KIF5–SNPH interaction mediates
activity-induced immobilization of
axonal mitochondria
Trak2 links mitochondrial receptor Miro1 to KIF5 motors to assemble mitochondrial transport complexes (MacAskill et al.,
354
JCB • VOLUME 202 • NUMBER 2 • 2013
2009a). Elevating Miro1 expression increases recruitment of
Trak2 and KIF5 to mitochondria, thus enhancing mitochondrial
transport (MacAskill et al., 2009b). Both Miro1 and Trak2 target axonal mitochondria (Fig. S2). Knocking down Trak2 or expressing its dominant-negative mutants reduces mitochondrial
motility in axons (Brickley and Stephenson, 2011). To determine whether Miro1/Trak2 and SNPH play opposite roles in the
regulation of axonal mitochondrial motility, we conducted six
panels of experiments in neurons. Expressing SNPH alone immobilized almost all axonal mitochondria (motility: 3 ± 1%),
whereas expressing Miro1 or Trak2 alone robustly increased
axonal mitochondrial motility (77 ± 3% [P < 0.001] and 81 ±
4% [P < 0.001], respectively) relative to control neurons (33 ±
2%). Interestingly, coexpressing SNPH and Miro1 or SNPH
and Trak2 dominantly immobilized axonal mitochondria (16 ±
2% [P < 0.001] and 14 ± 2% [P < 0.001], respectively; Fig. 3
and Videos 6 and 7).
The opposite effects of Miro1/Trak2 and SNPH in shifting
the balance between motile and stationary axonal mitochondria
raise two questions: whether (1) there is molecular interplay
between motors and docking receptors and (2) Miro-Ca2+ sensing immobilizes axonal mitochondria by switching the KIF5
interaction from motor adaptors to SNPH. To address these
questions, we performed six lines of biochemical analysis. First,
Downloaded from on June 12, 2017
snph/ neurons decreased significantly after the same treatments (P < 0.001; Fig. 1 E), consistent with the role of SNPH as
a docking receptor specific for axonal, but not dendritic, mitochondria (Kang et al., 2008).
We applied electrical field stimulation (100 Hz for 2 s) to increase firing rates and excitatory synaptic currents (MacAskill
et al., 2009b). Enhanced synaptic activity in snph+/+ neurons dramatically decreased axonal mitochondrial motility (37 ± 2%
before and 15 ± 3% after stimulation; P < 0.001). However, the
same treatment failed to change mitochondrial motility in snph/
neurons (77 ± 3% before and 73 ± 4% after stimulation; P = 0.10;
Fig. 2 and Videos 4 and 5), suggesting that deleting snph abolishes
activity-dependent regulation of axonal mitochondrial transport.
Removing extracellular Ca2 or blocking the action potential
using 1 µM tetrodotoxin (TTX), a sodium channel blocker, abolished mitochondrial immobilization in response to field stimulation in wild-type neurons (Fig. 2). Thus, SNPH is required to arrest
axonal mitochondrial movement in response to synaptic activity.
Published July 15, 2013
Downloaded from on June 12, 2017
Figure 4. SNPH interacts with KIF5. (A) GST-SNPH (203–469 aa) pulls down the kinesin-1 heavy chain (KHC or KIF5) from mouse brain homogenates
(BH). (B) An anti-SNPH antibody, but not preimmune serum (PIS), immunoprecipitates KHC from brain homogenates. KLC and the dynein intermediate
chain IC74 are not detected in the same assays. (C) Coimmunoprecipitation showing a native SNPH–KIF5 complex in mouse brains. SHPN was pulled
down by an anti-KHC antibody from wild-type (WT) mouse brain homogenates followed by detection with an anti-SNPH antibody. As a control, the same
antibody failed to detect the SHPN–KIF5 complex from snph knockout (KO) mouse brains. (D) GST pull-down showing SNPH binding to the C-terminal
tail (814–963 aa) of KIF5B. GST-SNPH (203–469 aa) or GST was incubated with His-tagged KIF5B truncated mutants. The bound protein complexes
were blotted with an anti–H7-His antibody. Coomassie blue staining shows the relative purity of the recombinant proteins. (E) Schematic diagram of
truncated SNPH mutants. MTB, MT binding domain; KBD, KHC binding domain; Mito, mitochondrial targeting sequence. (F) Coimmunoprecipitation
showing KIF5 binding to the SNPH sequence (381–466). GFP-KIF5C and GFP-SNPH truncated mutants were coexpressed in HEK293 cells. The cell
lysates were immunoprecipitated (IP) with an anti-KHC antibody and immunoblotted (IB) with an anti-GFP antibody. Note that the 86-residue segment
(381–466 aa) of SNPH is sufficient to bind to KIF5C and is thus referred to as the KBD. (G) Deleting KBD abolishes SNPH binding to KIF5C. GFPtagged KIF5C, SNPH, and its KBD deletion mutant, or KBD alone, were coexpressed in HEK293 cells. The cell lysates were immunoprecipitated with
an anti-KHC antibody and immunoblotted with an anti-GFP antibody. Red boxes indicate SNPH, its KBD deletion mutant, or KBD alone. Asterisks show
degraded bands.
we performed a pull-down assay using a truncated GST-SNPH
(203–469 aa), lacking the MT binding domain and Mito
(Fig. 4 E). GST-SNPH (203–469 aa) pulled down the kinesin-1
heavy chain (KHC), but not its light chain (KLC), and the
dynein intermediate chain (IC74) from mouse brain homogenates (Fig. 4 A). To confirm a native SNPH–KIF5 interaction
in neurons, we applied an anti-SNPH antibody to pull down endo­
genous KHC, but not KLC and IC74, from adult mouse brain
homogenates (Fig. 4 B). Conversely, SNPH was specifically
pulled down by the anti-KHC antibody from wild type, but not
from snph knockout, mouse brains (Fig. 4 C), providing convincing evidence for a native SNPH–KIF5 complex. Our pulldown and coimmunoprecipitation assays suggest that SNPH
interacts with KIF5 independent of KLC. In contrast, a native
SNPH–dynein heavy chain (DHC) complex was not detected
by either an anti-SNPH or an anti-DHC antibody from mouse
brains under the same conditions (Fig. S3).
Next, we examined the direct interaction between SNPH
and KHC. Mammals have three KHCs (KIF5A, -B, and -C), which
share a highly conserved sequence homology, particularly in
their C-terminal cargo binding domains (Hirokawa et al., 2010).
SNPH specifically binds His-KIF5B (814–963 aa), a C-terminal
tail containing the cargo binding domain (Fig. 4 D and Fig. S4).
In contrast, no SNPH binding was observed for His-KIF5B
(1–554 aa), the N-terminal motor domain (N-MD), or HisKIF5B (555–813 aa), the middle stalk coils. Furthermore, we
located a KHC binding sequence in SNPH by performing coimmunoprecipitation in HEK293 cells coexpressing GFP-tagged
KIF5C, a neuron-specific Kinesin-1 isoform, and various truncated SNPH mutants. The 86-residue segment (381–466 aa) of
Regulation of axonal mitochondrial transport • Chen and Sheng
355
Published July 15, 2013
SNPH is sufficient to bind to KIF5C and is hereafter referred
to as KHC binding domain (KBD; Fig. 4, E and F). In addition,
we generated an SNPH mutant lacking KBD (SNPH-KBD).
Although KBD alone is sufficient to interact with KIF5C, no
binding was detected for SNPH-KBD to KIF5C (Fig. 4 G), suggesting direct interaction between SNPH and KIF5 via KBD.
Next, we tested whether KIF5–SNPH interaction is required to immobilize axonal mitochondria. Although GFPSNPH arrested almost all axonal mitochondria (Fig. 5, A and B),
a significant portion (24%) of mitochondria labeled with
SNPH-KBD moved dynamically. Given the fact that SNPHKBD contains the MT binding domain (103–203 aa) and the
Mito (466–532 aa; Fig. 4 E), overexpressing this mutant on
mitochondria would enhance the static link between mitochondria and MTs. We alternatively performed immunoprecipitation
using cell lysates from transfected HEK cells. Although the
KIF5C–SNPH complex was readily detectable in the absence
of expression of KBD, coexpressing KBD reduced the KIF5C–
SNPH complex (Fig. 5 C), suggesting that KBD interferes
with the KIF5–SNPH interaction. To validate the biochemical
observations, we performed live-neuron imaging. Field stimulation (100 Hz for 2 s) of neurons expressing KBD failed to arrest axonal mitochondrial transport (40 ± 2% before and 37 ±
4% after stimulation; P = 0.30; Fig. 5, D and E; and Video 8).
However, the same stimulation in neurons expressing GFP control results in a rapid reduction in mitochondrial movement
356
JCB • VOLUME 202 • NUMBER 2 • 2013
(34 ± 2% before and 14 ± 2% after stimulation; P < 0.001).
In contrast, expressing KBD did not impair activity-dependent
immobilization of dendritic mitochondria (32 ± 9% before and
6 ± 5% after stimulation; P < 0.001). These results suggest that
KIF5–SNPH coupling is crucial for activity-dependent immobilization of axonal but not dendritic mitochondria.
Miro-Ca2+ sensing facilitates
KIF5–SNPH interaction
We then examined whether the KIF5–SNPH interaction has
any impact on the complex KIF5–Trak2–Miro1. First, we coexpressed GFP-KIF5C and Flag-Trak2 in the absence or presence of SNPH or SNPH-KBD in HEK293 cells followed
by immunoprecipitation with an anti-Flag antibody. Although
KIF5C–Trak2 complexes were readily detectable in the absence
of SNPH, coexpressing SNPH, but not SNPH-KBD, reduced
KIF5C–Trak2 complexes (Fig. 6, A and B). As the KIF5C
C-terminal tail binds both Trak2 (Smith et al., 2006) and SNPH,
it is likely that SNPH and Trak2 competitively bind KIF5C. Second, expressing Trak2 alone in neurons significantly increased
axonal mitochondrial motility (81 ± 4%) relative to wild-type
control (P < 0.001). When coexpressing Trak2 with SNPH, only
a small portion (14 ± 2%) of axonal mitochondria was motile. In
contrast, when coexpressing Trak2 with SNPH-KBD, a larger
portion (50 ± 3%; P < 0.001) of axonal mitochondria was motile
(Fig. 6, C and D). Based on the in vitro biochemical analyses
Downloaded from on June 12, 2017
Figure 5. Disrupting KIF5–SNPH interaction abolishes activity-dependent immobilization of axonal mitochondria. (A and B) Kymographs (A) and quantitative analysis (B) showing the motility of axonal mitochondria in neurons expressing GFP-SNPH or GFP-SNPH-KBD. (C) An inhibitory role of KBD
in the KIF5C–SNPH interaction. HEK293 cells were transfected with GFP-KIF5C alone or cotransfected with GFP-SNPH or GFP-SNPH + GFP-KBD. The
KIF5C–SNPH complex was immunoprecipitated (IP) from the lysates using an anti-KHC antibody followed by immunoblotting (IB) with an anti-GFP antibody.
Note that KBD interferes with the interaction of SNPH with KIF5C (top red box) by competitively binding to KIF5C (bottom red box). (D and E) The motility
of axonal and dendritic mitochondria in wild-type neurons before and after 100-Hz (2 s) field stimulation. Expressing GFP-KBD abolishes the activity-dependent
immobilization of axonal but not dendritic mitochondria (also see Video 8). Data were quantified from numbers of neurons (indicated in parentheses in
B and within bars in E) in three independent experiments. Bars, 10 µm. Error bars show SEM. Student’s t test.
Published July 15, 2013
Downloaded from on June 12, 2017
Figure 6. SNPH competes with Trak2 to bind KIF5. (A and B) Representative blots (A) and quantitative analysis (B) showing competition of SNPH and Trak2
for binding KIF5. HEK293 cells were cotransfected as indicated. The KIF5C–Trak2 complex was coimmunoprecipitated (IP) from cell lysates with an antiFlag antibody and immunoblotted (IB) with an anti-GFP antibody (bottom). Note that SNPH, but not SNPH-KBD, inhibits the formation of the KIF5C–Trak2
complex (red box). Data were quantified from three independent experiments. The asterisk denotes degraded GFP-SNPH. (C and D) SNPH, but not SNPHKBD, competes with Trak2 to immobilize axonal mitochondria. (E and F) Immunoprecipitation (E) and quantitative analysis (F) showing activity-induced
and Ca2+-dependent formation of the native SNPH–KHC complex in mature cortical neurons. The cultured neurons (DIV14) were treated for 10 min with
50 mM KCl and 10 µM FPL 64176 in modified Tyrode’s solution with or without 1.2 mM Ca2+. The native SNPH–KHC complexes were coimmunoprecipitated from neuronal lysates with an anti-SNPH antibody and subsequently immunoblotted with an anti-KHC and LC8 antibody. Coimmunoprecipitated
levels of KIF5 and LC8 after stimulation were normalized against their levels before stimulation, respectively. Note that removing [Ca2+]ext from the medium
abolished the activity-induced SNPH–KIF5 interaction. (G and H) Kymographs (G) and quantitative analysis (H) showing enhanced motility of axonal mitochondria after Ca2+ depletion. Mitochondrial motility in hippocampal neurons were recorded before and 20 min after removing 1.2 mM [Ca2+]ext from the
medium on the same axons (also see Video 9). Bars, 10 µm. Data in D and H were quantified from numbers of axons (indicated in bars). Student’s t test
in D and H and Mann–Whitney U test in B and F. Error bars show SEM.
in soluble conditions and live-neuron imaging, we hypothesize
that SNPH-mediated regulation of axonal mitochondrial transport is likely by competing with Trak2 for binding KIF5.
Next, we asked whether elevated Ca2+ levels facilitate formation of the native SNPH–KIF5 complex in mature cortical
neurons (day in vitro [DIV] 14) treated for 10 min with 50 mM
KCl and 10 µM FPL 64176 in the presence or absence of 1.2 mM
Ca2+ in modified Tyrode’s solution. Removing [Ca2+]ext from
the medium abolished the activity-enhanced SNPH–KIF5 inter­
action (Fig. 6, E and F). As an internal control, neuronal activity
has no influence on the SNPH-LC8 binding. We further examined the relative motility of axonal mitochondria under the resting conditions in the absence of [Ca2+]ext. Mitochondrial motility
was recorded before and 20 min after removing [Ca2+]ext from
medium on the same axons. In the resting state, one third (33 ±
3%) of axonal mitochondria were motile in the presence of 1.2 mM
[Ca2+]ext. Removing [Ca2+]ext from the medium significantly increased the motility (56 ± 3%; P < 0.001; Fig. 6, G and H; and
Video 9), supporting the notion that the SNPH–KIF5 inter­action
is induced by Ca2+. Depleting Ca2+ facilitates the dissociation of
SNPH from KIF5 motors, thus relieving the inhibitory role of
SHPN on the motor activity.
SNPH–KIF5 coupling inhibits motor activity
Although two thirds of axonal mitochondria are in the stationary phase, deleting snph in mice results in >70% of axonal
Regulation of axonal mitochondrial transport • Chen and Sheng
357
Published July 15, 2013
mitochondria in the motile state (Kang et al., 2008). To address
whether this robustly enhanced motility is caused by increased
recruitment of molecular motors to mitochondria, we isolated
axonal mitochondria from sciatic nerves of snph+/+ and snph/
mice as previously described (Frezza et al., 2007). Immuno­
blot analysis showed no significant change in the normalized
intensity of axonal mitochondria-associated KIF5 (P = 0.28)
and dynein intermediate chain IC74 (P = 0.88) between snph+/+
and snph/ mice (Fig. 7, A and B), suggesting that KIF5 and
358
JCB • VOLUME 202 • NUMBER 2 • 2013
Downloaded from on June 12, 2017
Figure 7. SNPH inhibits KIF5 motor ATPase activity. (A and B)
Deleting snph has no effect on the motor association with axonal mitochondria. Axonal mitochondria were isolated from
sciatic nerves of snph+/+ and snph/ mice. 15 µg of total
proteins from supernatant and mitochondria-enriched fractions
was loaded in SDS-PAGE and sequentially immunoblotted on
the same membrane after stripping between each antibody
application. The intensities of KIF5 and dynein IC74 were
normalized to mitochondrial marker Tom20. Relative purity
of the mitochondrial fraction was assessed by sequential immunoblotting mitochondrial marker Tom20, SNPH, and other
markers including EEA1 (endosomes), Grp78 (ER), and actin.
Data were collected from four paired littermates. Error bars
show SEM; Mann–Whitney U test. (C) SNPH and Trak2 play
opposite roles in regulating KIF5C ATPase. ATPase activity
of full-length KIF5C or its motor domain (KIF5C–N-MD) was
measured using Kinesin ATPase Endpoint Biochem kit and expressed as hydrolyzed ATP. MT-stimulated ATPase activity of
both KIF5C (blue) and KIF5C–N-MD (green) was used as a reference value for subsequent endpoint assays by adding SNPH
(or its mutant) or Trak2 under the same conditions. Incubating SNPH (1–469 aa) significantly reduces the motor ATPase
activity (P = 0.029), whereas Trak2 increases the activity
(P = 0.028). Deleting the KIF5 binding domain (KBD) abolishes its inhibitory role. Error bars show SEM. Mann–Whitney
U test, n = 4. (D) Representative time-lapse imaging showing
the movement of EGFP-KIF5C along MTs labeled with mCherry–
-tubulin in a live COS cell. (E) Mean velocity of EGFP-KIF5C
puncta along MTs in living cells with or without expression
of SNPH, its KBD mutant, or Trak2. Note that coexpressing
SNPH immobilizes KIF5C puncta. Data were quantified from a
total number of EGFP-KIF5C puncta (indicated in parentheses
above the bars). Error bars show SEM. Student’s t test. WT,
wild type; KO, knockout.
dynein motors attach to both motile and stationary mito­chondria.
This study raises a question as to whether motile mito­chondria
are recruited to the stationary pool by inactivating motors
on mitochondria.
KIF5 motors undergo a mechanochemical cycle by hydrolyzing ATP into ADP and Pi; the energy released from the phosphate drives motor translocation along the MT filament. The
N-terminal domain of KIF5 is comprised of a motor domain
with ATPase and MT-binding activity (Vale, 2003; Hirokawa
Published July 15, 2013
SNPH is recruited to axonal mitochondria
in response to neuronal activity
We then examined whether SNPH-labeled mitochondria are
recruited to presynaptic boutons in response to synaptic activity. Colocalization of SNPH-labeled mitochondria with
synaptic marker synaptophysin was increased after the KCl
treatment (P < 0.001; Fig. 8, A and B), supporting the hypothesis that action potential firing recruits axonal mitochondria to synapses. SNPH was recruited to axonal mitochondria
after a 100-Hz field stimulation. Before stimulation, 24 ± 2%
of axonal mitochondria display low levels of SNPH staining
(defined as SNPH/cytochrome c intensity ratio <0.5). These
low levels of SNPH were only visible when the image contrast
was enhanced. This staining pattern is consistent with our previous study that under regular imaging contrast settings, one
third of axonal mitochondria had no detectable SNPH staining (Kang et al., 2008). Sustained neuronal activity increased
SNPH intensity on axonal mitochondria (Fig. 8, C and D).
These data suggest that (a) SNPH enrichment on axonal mitochondria is heterogeneous, and (b) SNPH undergoes dynamic
recruitment to axonal mitochondria in response to neuronal
activity although the underlying mechanism directing SNPH
translocation is unclear.
Discussion
KIF5–SNPH coupling turns off “engine
switch” to immobilize mitochondria
Recent studies characterized Miro’s role as a Ca2+ sensor
for activity-dependent regulation of mitochondrial transport
(Saotome et al., 2008; MacAskill et al., 2009b; Wang and
Schwarz, 2009; Sheng and Cai, 2012). In the current study,
we demonstrate that SNPH mediates the activity-dependent
immobilization of mitochondria in axons. The SNPH–KIF5
coupling is triggered by a Miro-Ca2+ sensor, which releases
the C-terminal tail of KIF5 to bind SNPH, resulting in inhibition of the motor ATPase. Thus, SNPH arrests axonal mitochondrial movement in response to synaptic activity by (a)
interfering with the motor transport complex and (b) inactivating the motor ATPase. Deleting the snph gene abolishes
the activity-induced immobilization of mitochondria in axons
but not in dendrites. Here, we propose a new “Engine-Switch
and Brake” model (Fig. 8 E), in which SNPH acts both as an
engine off switch by sensing stop sign-Ca2+ and as a brake by
anchoring mitochondria to the MT track. When in their stationary phase, both the KIF5 motor and mitochondria remain
bound to the MT track.
Our model nicely reconciles the standing dispute regarding how Miro-Ca2+ sensing arrests mitochondrial transport.
MacAskill et al. (2009b) suggested that Ca2+ binding to Miro
releases KIF5 motors from mitochondria. Glutamate application led to a 45% decrease in the amount of KIF5 motors bound
to mitochondria. Because glutamate acts predominantly on
dendrites where SNPH is absent, glutamate evokes immobilization of dendritic mitochondria likely via a Ca2+-dependent
dissociation of KIF5 from mitochondria. Alternatively, Wang
and Schwarz (2009) found that in axons, KIF5 remained associated with stationary and motile mitochondria at both resting
and elevated Ca2+ conditions. These discrepant findings may
account for their selective observations of mitochondrial motility in axons versus dendrites. SNPH is an axonal mitochondrial outer membrane protein (Kang et al., 2008). Thus, upon
Miro-Ca2+ sensing, KIF5 motors remain on axonal but not
dendritic mitochondria, likely through its coupling to SNPH.
Our findings suggest that motor–cargo association is insufficient for the motor-driven transport, consistent with a recent
in vivo study on prion protein vesicles, which showed that
both KIF5 and dynein motors remain bound to organelles regardless of whether they were moving or stationary (Encalada
et al., 2011).
One third of axonal mitochondria displayed low levels or
undetectable SNPH (Kang et al., 2008). Sustained neuronal activity may recruit more SNPH to axonal mitochondria. Synaptic
localization of mitochondria in snph/ hippocampal neurons
was significantly lower (P < 0.001). Thus, during prolonged
stimulation in the snph mutant neurons, short-term facilitation
is enhanced as a result of impaired [Ca2+] buffering at presynaptic terminals. This phenotype is fully rescued by reintroducing the snph gene into the mutant neurons (Kang et al., 2008).
One of remaining questions is how stationary mitochondria
move again. The sequence features of SNPH, 12% residues as
Regulation of axonal mitochondrial transport • Chen and Sheng
Downloaded from on June 12, 2017
et al., 2010). We next examined whether the binding of SNPH
or Trak2 to the KIF5 tail could have any impact on its motor
ATPase activity. First, we performed endpoint ATPase activity assays as previously described (Cho et al., 2009). Flagtagged full-length KIF5C or its N-MD (KIF5–N-MD) was
purified with anti-Flag magnetic beads and incubated with
SNPH or its mutants or Trak2 in the presence or absence of
MTs (Fig. 7 C). MT-stimulated ATPase activity was used as a
reference value. Coincubating SNPH (1–469 aa), which contains the KBD, significantly reduces KIF5C ATPase activity
(P = 0.029), whereas Trak2 plays an opposite role by increasing the motor ATPase activity (P = 0.028) under the same conditions. Deleting the KBD (KBD) abolishes its inhibitory
role (P = 0.147). In contrast, coincubating SNPH or Trak2
has no significant effect on ATPase activity of KIF5–N-MD,
which lacks the binding domains for SNPH and Trak2. Thus,
by binding to the C-terminal domain of KIF5, the docking
adaptor SNPH inhibits motor activity while Trak2 stimulates
the motor ATPase.
To evaluate the functional role of SNPH in inactivating KIF5 motors, we examined the velocity of KIF5 along
MT tracks in live COS cells expressing EGFP-KIF5C alone
or co­expressing with SNPH. A previous study characterized
movements of vesicular EGFP-KIF5C structures along MTs
in COS cells with a velocity similar to that measured in vivo
(Dunn et al., 2008). Our live-imaging analysis showed motile EGFP-KIF5C vesicular structures along MTs labeled
with mCherry–-tubulin (Fig. 7 D). The mean velocity of
the motor puncta in cells expressing KIF5C alone is 0.47 ±
0.05 µm/s. However, coexpressing SNPH, but not SNPHKBD, immobilized KIF5C puncta (0.03 ± 0.01 µm/s; P < 0.001),
further supporting our hypothesis that SNPH inactivates KIF5
motor function.
359
Published July 15, 2013
Downloaded from on June 12, 2017
Figure 8. SNPH is recruited to axonal mitochondria in response to neuronal activity. (A and B) Representative images (A) and quantitative analysis (B)
showing the relative colocalization of SNPH-labeled axonal mitochondria with synapses. Neurons before and after the KCl treatment were fixed and
coimmunostained with antibodies against SNPH and the synaptic marker synaptophysin (synapto). Note that action potential firing recruits SNPHlabeled mitochondria to synapses. Data were quantified from a total number of neurons (indicated within the bars) in three independent experiments.
(C and D) Representative images (C) and quantitative analysis (D) showing increased SNPH enrichment into axonal mitochondria after 100-Hz field
stimulation. Cultured hippocampal neurons were coimmunostained with anti-SNPH and anti–cytochrome c (cyto c). Arrows point to axonal mitochondria
with low or no SNPH staining. Mean SNPH intensity on each mitochondrion was normalized against cytochrome c intensity. Relative SNPH enrichment on
mitochondria was expressed as an intensity ratio (SNPH/cytochrome c; D, top) and the percentage of low SNPH-labeled axonal mitochondria (defined by
the SNPH/cytochrome c ratio <0.5; D, bottom). Note that those tiny SNPH puncta not colabeled with mitochondria may represent shuttle cargoes delivering
SNPH from axoplasm to mitochondria. Data were quantified from several axonal mitochondria (M) from the total number of neurons (N) examined in three
independent experiments. Bars, 10 µm. Error bars show SEM. Student’s t test. (E) Proposed “Engine-Switch and Brake” model. SNPH plays a critical role
in docking mitochondria via directly binding KIF5 and inhibiting motor ATPase activity. KIF5–SNPH molecular interplay is triggered by a Miro-Ca2+ sensing that releases KIF5 to bind to SNPH. Thus, SNPH turns off the “engine” (KIF5 motor) by sensing a “stop sign” (elevated Ca2+) and arrests mitochondria
moving along the MT track. When in their stationary phase, mitochondria remain bound to the MT tracks, whereas KIF5 is in an inactive state.
360
JCB • VOLUME 202 • NUMBER 2 • 2013
Published July 15, 2013
serine or threonine and numerous phosphorylation sites, suggest that its role as a brake could be turned off through signal
transduction pathways. Given that SNPH is a cellular target of
the E3 ubiquitin ligase Cullin 1 (Yen and Elledge, 2008), mitochondria-targeted SNPH may also be terminated by ubiquitinmediated degradation pathways.
SNPH arrests bidirectional transport
of axonal mitochondria
SNPH-mediated mitochondrial
immobilization and synaptic transmission
Mitochondria maintain synaptic transmission by producing
ATP and maintaining Ca2+ homeostasis at synapses (Tang and
Zucker, 1997; Kang et al., 2008). SNPH was originally isolated
through a yeast two-hybrid screening using the coiled-coil domain of syntaxin-1A as bait (Lao et al., 2000). SNPH coiledcoil domain (130–203 aa) shares 80% structural similarity
to the syntaxin coiled-coil domain. Earlier studies showed that
SNPH plays a role in reducing synaptic transmission after acute
overexpression (6–12 h) of recombinant SNPH in autaptic hippocampal neurons through Semliki Forest virus infection or after
Downloaded from on June 12, 2017
The Miro-Ca2+ sensing pathway equally affects both anterograde and retrograde mitochondrial transport (Cai and Sheng,
2009; MacAskill et al., 2009b; Wang and Schwarz, 2009),
leaving a question as how the KIF5 adaptor complex affects
dynein-driven retrograde transport. Dynein has been observed
colocalizing with mitochondria moving in both the anterograde
and retrograde directions (Hirokawa et al., 1990). Inhibiting
kinesin-1 in Drosophila also reduces retrograde mitochondrial
movement (Pilling et al., 2006). Thus, the kinesin and dynein
motors may coordinate bidirectional transport rather than simply
competing against one another (Ligon et al., 2004). Any change
in this coordination may alter the motor processivity, time spent
in stationary phases, run lengths, or pause frequencies (Gross
et al., 2007; Encalada et al., 2011). Miro is one candidate for
regulating bidirectional transport. dMiro in Drosophila regulates both anterograde and retrograde mitochondrial transport
(Russo et al., 2009). Mitochondrial fusion proteins mitofusins
(Mfn1 and Mfn2) interact with Miro, and this interaction is
necessary to regulate axonal mitochondrial transport. Deleting
Mfn2 or expressing Mfn2 mutants results in axonal mitochondria spending more time paused and undergoing slower anterograde and retrograde transport (Misko et al., 2010).
Identifying SNPH as a motor “brake” provides new mechanistic insights into how Miro-Ca2+ sensing arrests mitochondrial
bidirectional movement. Miro-Trak2 and SNPH can displace
one another when binding to the KIF5 motor, and SNPH–KIF5
coupling inhibits the motor ATPase. Deleting snph impairs
the motor brake, thus mitochondria are unable to be immobilized upon the Miro-Ca2+ sensing. Given the fact that no native SNPH–dynein complex is detected in mouse brains, SNPH
may affect retrograde transport through inhibiting KIF5 motor.
Our model is similar to a general “Molecular Brakes” model
(Gross et al., 2007), in which the cargo-MT linkage via specific
“coupling proteins” could act as molecular brakes to stop bidirectional movement.
microinjection of highly concentrated coiled-coil domain (50 mg)
into superior cervical ganglion neurons (Lao et al., 2000). It is
likely that there is a competitive interference between the SNPH
coiled-coil domain and the SNARE machinery. We further characterized the snph knockout mice combined with gene rescue
experiments and found that deleting snph had no significant
impact on mini-AMPA events and basal synaptic transmission.
However, deleting snph results in increased presynaptic [Ca2+]
levels during prolonged high-frequency stimulation (Kang et al.,
2008). This phenotype is attributed to fewer mitochondria being
tethered at terminals, thus reducing the Ca2+ buffering capacity.
In the same snph/ neurons, dendritic mitochondria are immobilized in response to neuronal activity as efficiently as in the
wild-type neurons. Therefore, the defects in activity-dependent
immobilization of axonal mitochondria are less likely a result
of adaptation of the mutant neurons to Ca2+ influx, lack of appropriate receptors, or downstream signaling molecules.
Defective mitochondrial trafficking was implicated in the
pathological processes of major neurodegenerative diseases
(Sheng and Cai, 2012). Selective mitochondrial degradation
through mitophagy plays an important role in the quality control of mitochondria (Chen and Chan, 2009; Cai et al., 2012).
Whether and how altered mitochondrial motility impacts the
turnover of dysfunctional mitochondria in axons and at terminals are unclear. Our current findings provide molecular targets
for future studies investigating into how disease-related mutations impair mitochondrial transport and docking machineries.
These studies may advance our understanding of human neurodegenerative disorders.
Materials and methods
Mouse line
The snph/ mouse line was generated by targeted gene replacement in
embryonic stem (ES) cells as previously described (Kang et al., 2008). All
four coding exons of the snph gene were replaced by the selection cassette
phosphoglycerate kinase (PGK) neo (neomycin resistance gene under the
PGK promoter). The herpes simplex virus-1 thymidine kinase gene under
the PGK promoter (thymidine kinase cassette) was placed at the end of the
long arm of the construct. The vector was linearized and electroporated
into ES cell line TC1 (derived from a 129/SvEvTacfBR mouse) and then selected with G418 and fialuridine. Homologous recombination events were
identified by Southern blot screening using a PCR-generated probe. ES cells
heterozygous for the targeted mutation were independently microinjected
into C57BL/6J blastocysts, generating chimeric mice that transmitted the
mutation through the germline (germline transmission). Heterozygous and
knockout mice were generated and identified by Southern blotting and PCR
amplification. Both homozygote and heterozygote mutant animals were viable, fertile, and morphologically normal. Animal care and use were performed in accordance with National Institutes of Health guidelines and
were approved by the National Institutes of Health National Institute of
Neurological Disorders and Stroke/National Institute on Deafness and
Other Communication Disorders Animal Care and Use Committee.
DNA constructs
The following DNA constructs were gifts: pRK5Myc-Miro1 obtained from
P. Aspenström (Karolinska Institutet, Stockholm, Sweden); pEGFP-KIF5C
obtained from M. Peckham (University of Leeds, Leeds, England, UK);
pCMVFlag-Trak2 obtained from F.A. Stephenson (University College London, London, England, UK); and pET-28a-KHC truncated mutants obtained
from R.J. Diefenbach (The University of Sydney and Westmead Hospital,
Westmead, Australia). Various truncated SNPH mutants were generated by
PCR and subcloned into pEGFP-C and pGEX-4T vectors (GE Healthcare).
An SNPH (381–466 aa) deletion was introduced by mutagenesis (Quik­
Change; Agilent Technologies). The full-length coding sequences of Miro1
Regulation of axonal mitochondrial transport • Chen and Sheng
361
Published July 15, 2013
and Trak2 were cloned by PCR and inserted into the pEGFP-C vector.
pEYFP-tubulin containing full-length human -tubulin was purchased from
Takara Bio Inc. and subcloned into pmCherry-C1 vectors.
Antibodies and reagents
The purified rabbit polyclonal antibody against SNPH residues 225–428
was described previously (Kang et al., 2008). Sources of other antibodies
or reagents are as follows: monoclonal anti-KIF5 (heavy chain) and anti–
dynein IC74 antibodies (EMD Millipore); polyclonal anti-Flag, monoclonal
anti-actin, and anti–-tubulin (Sigma-Aldrich); monoclonal anti–cytochrome
c (BD); polyclonal anti-EEA1, anti-TOM20, anti-KLC, anti-DHC, and monoclonal anti-synaptophysin (Santa Cruz Biotechnology, Inc.); monoclonal
anti-GFP (JL-8; Takara Bio Inc.); ECL-HRP–linked secondary antibodies (GE
Healthcare); Alexa fluor 546– or 633–conjugated secondary antibodies
(Invitrogen); Pepstatin A (EMD Millipore); and leupeptin (Sigma-Aldrich).
COS and HEK293 cell cultures and transfection
COS and HEK293 cells were cultured in high-glucose DMEM containing
sodium pyruvate and l-glutamine supplemented with 10% FBS and penicillin–
streptomycin (Invitrogen) until the cells became confluent. Transient transfection
was performed using Lipofectamine 2000 (Invitrogen). After transfection, cells
were cultured for an additional 1–2 d before immunocytochemistry or harvesting for biochemical analysis.
Immunocytochemistry
Cultured cells were fixed with 4% paraformaldehyde and 4% sucrose at RT
for 15 min, washed three times with PBS for 5 min each, permeabilized in
0.1% Triton X-100 for 15 min, and then incubated in 5% normal goat
serum in PBS for 1 h. Fixed cells were incubated with primary antibodies
in PBS with 1% BSA at 4°C overnight or at RT for 4 h. Cells were washed
four times with PBS at RT for 5 min each, incubated with secondary fluorescent antibodies in PBS with 1% BSA for 30 min, rewashed with PBS, and
mounted with antifade mounting medium (Fluoro-Gel; Electron Microscopy
Sciences) for imaging.
Recombinant protein preparation
GST-tagged proteins were expressed in Escherichia coli BL21 (DE3). The
BL21 cells were grown to an OD600 of 0.6; expressed protein was induced
with IPTG to a final concentration of 0.5 mM at 30°C for 4–6 h. The cell
pellet was resuspended in 20 mM phosphate buffer, pH 7.6, containing
150 mM NaCl, 0.5 mM DTT, and protease inhibitors (1 mM phenylmethylsulfonyl fluoride, 0.1 mM benzamidine, and 3 µg/ml each of leupeptin
and pepstatin). After sonication and centrifugation, the crude extract was
purified using GST–Sepharose beads (GE Healthcare). Expression of HisKIF5C and its truncated fragments were purified with Ni–nitrilotriacetic
acid beads (QIAGEN) according to the manufacturer’s instructions.
GST pull-down assay
Rat brains were homogenized in buffer A (25 mM Tris-HCl, pH 7.5, 50 mM
NaCl, 1 mM EDTA, 1 mM DTT, 0.5% Triton X-100, and protease inhibitors
including 1 mM phenylmethylsulfonyl fluoride, 0.1 mM benzamidine, and
3 µg/ml each of leupeptin and pepstatin). The crude homogenate was centrifuged at 15,000 g for 40 min; the supernatant was carefully collected.
GST beads coated with 4 µg GST, GST-SNPH, or its truncated mutants
were mixed with the brain homogenates and incubated for 3 h with gentle
362
JCB • VOLUME 202 • NUMBER 2 • 2013
In vitro binding assay and coimmunoprecipitation
10 µl glutathione–Sepharose resin were added to 4 µg of GST-SNPH
or its truncated and deleted mutants; the mixture was incubated on ice
for 30 min before adding 8 µg purified His-KIF5C. The mixture was
incubated on ice for another 1–3 h. The resin was then washed four
times. Bound protein was eluted from the resin with 30 µl of 2× SDS
sample buffer and heated at 95°C for 10 min. After brief centrifugation, the supernatant was subjected to SDS-PAGE followed by Western
blot analysis.
Mouse brains or HEK293 cells or cultured cortical neurons were
homogenized in TBS with 1% Triton X-100 and protease inhibitors (1 mM
phenylmethylsulfonyl fluoride, 0.1 mM benzamidine, and 3 µg/ml each of
leupeptin and pepstatin). The crude lysates were centrifuged at 13,000 g
for 30 min, and the supernatant was collected. The lysates were incubated
with 3 µg antibody or preimmune serum in 0.5 ml TBS with 0.1% Triton
X-100 and protease inhibitors and incubated on a rotator at 4°C for 2 h.
Protein A–Sepharose CL-4B resin (GE Healthcare) was added to each sample; incubation continued for an additional 3 h followed by three washes
with TBS/0.1% Triton X-100. The beads were dissolved in SDS-PAGE sample buffer and heated at 95°C for 10 min. The proteins were resolved by
SDS-PAGE and processed for Western blot analysis. For semiquantitative
analysis, protein bands detected by ECL were scanned into Photoshop
CS3 (Adobe) and analyzed using protein imaging software (Gel-Pro 4.5;
Media Cybernetics). Care was taken during exposure of the ECL film to
ensure that intensity readouts were in a linear range of standard curve blot
detected by the same antibody.
Analysis of mitochondrial motility and imaging data acquisition
Both axonal and dendritic processes were selected for mitochondrial motility analysis. As we previously reported (Kang et al., 2008; Cai et al.,
2010), axons in live images were distinguished from dendrites based
on known morphological characteristics: greater length, thin and uniform
diameter, and sparse branching (Banker and Cowan, 1979). In addition,
axonal mitochondria are more sparsely distributed along axons and display distinct morphology patterns as small, vesicular, or short vesicular–tubular
structures. In contrast, dendritic mitochondria are elongated, tubular, or filamentous structures occupying a large fraction of dendritic processes
(Popov et al., 2005; Chang et al., 2006). We selected the proximal region
of axons and dendrites for time-lapse imaging analysis. Only those that
appeared to be single axons and separate from other processes in the field
were chosen for recording axonal mitochondrial transport. Regions where
crossing or fasciculation occurred were excluded from analysis. Axonal
processes were further confirmed by retrospective immunostaining with an
anti-MAP2 antibody.
Time-lapse imaging was performed at 37°C using a confocal microscope (LSM 510 META; Carl Zeiss) with a C-Apochromat 40×/1.2 NA
water Corr objective (Carl Zeiss) and a perfusion system (0.4 ml/min) with
modified Tyrode’s solution (10 mM Hepes, 10 mM glucose, 3 mM KCl, 145
mM NaCl, 1.2 mM CaCl2, 1.2 mM MgCl2, and 1 µM glycine, pH 7.4).
Time-lapse images were collected at a 512 × 512–pixel resolution (8 bit).
A relatively long interval (10 s) for a total of 100 images was used to minimize laser-induced cellular damage. Kymographs were made with extra
plug-ins for ImageJ (National Institutes of Health) as previously described
(Kang et al., 2008). In brief, we used plug-ins “LSM_Reader” to read Carl
Zeiss LSM images, “Straighten” to straighten curved axons, “Grouped_
ZProjector” to z-axially project resliced time-lapse images, “Time_Stamper”
to make time-stamped images/frames, and “QT_Movie_Writer” to make
the video files as MOV format. The height of the kymographs represents
recording time, and the width of the kymographs represents length (micro­
meters) of the axon imaged. A mitochondrion was considered stationary
if it remained immotile for the entire recording period; a motile one was
counted only if the displacement was ≥5 µm.
Electric field stimulation
During time-lapse imaging, cultured hippocampal neurons transfected
with DsRed-Mito were treated with 100-Hz field stimulation, which was
Downloaded from on June 12, 2017
Hippocampal neuronal culture and transfection
Hippocampi were dissected from postnatal day 1 mouse or rat pups and
cultured as previously described (Ilangovan et al., 2005). In brief, after
dissociation with papain (Worthington), hippocampal cells recovered by
centrifugation were plated onto 12-mm coverslips in 2 ml of plating medium in 35-mm dishes (for 10 ml of plating medium: 9.5 ml Neurobasal
medium [Invitrogen], 0.5 mM glutamine, 0.25 mg insulin, 5% fetal bovine
serum, 2% B-27 medium supplement, and G-5 supplement). Beginning on
the second day in culture, half of the medium was replaced with feeding
medium twice a week (10 ml of feeding medium: 9.8 ml Neurobasal medium and 2% B-27 medium supplement). To label mitochondria and early
and late endosomes, DsRed-Mito and YFP-Rab7 or GFP-Rab5 constructs
were cotransfected into neurons at DIV9 using the calcium phosphate
method (Jiang and Chen, 2006). In brief, the DNA-Ca2+ and phosphate solutions were gently mixed and incubated at room temperature for 20 min.
After incubation with cultured neurons on the coverslip for 45 min, the
coverslip was washed with prewarmed DMEM and transferred to a new
dish containing original culture medium followed by time-lapse imaging in
live neurons 3–4 d after transfection.
rotation. The beads were then extensively washed with buffer B (25 mM
Tris-HCl, pH 7.5, 100 mM NaCl, 1 mM DTT, 1 mM EDTA, 0.1% Nonidet
P-40, and protease inhibitors). The beads were dissolved in SDS-PAGE
sample buffer and heated at 95°C for 10 min. Proteins were resolved by
SDS-PAGE and processed for Western blot analysis. For multiple detections
with different antibodies on the same membranes, blots were stripped in a
solution of 62.5 mM Tris-HCl, pH 7.5, 20 mM DTT, and 1% SDS for 20 min
at 50°C with agitation and washed twice with TBS/0.1% Tween 20 for
15 min each time.
Published July 15, 2013
generated with a stimulator (Master 8; A.M.P.I.) and amplified with stimulus isolation (A385; World Precision Instruments). 200 biphasic pulses
were delivered through platinum electrodes positioned on opposite sides
of the field stimulation chamber (Warner Instruments) at 1 ms of stimulus
duration and 50 mA of constant current. 1.2 mM CaCl2 was removed
from Tyrode’s solution for control experiments in the absence of extracellular calcium. 1 µm TTX was added to Tyrode’s solution for the action
potential blocking experiment.
Preparation of axonal mitochondria-enriched membrane fractions
Axonal mitochondria were prepared from sciatic nerves of 9-month-old
snph+/+ and snph/ mice as previously described (Frezza et al., 2007).
Mouse sciatic nerves were homogenized in mitochondria isolation buffer
(10 mM Tris-MOPS, 1 mM EGTA, and 20 mM sucrose plus protease inhibitors, pH 7.4) centrifuged at 600 g for 10 min at 4°C. The supernatants
were collected and further centrifuged at 7,000 g for 10 min at 4°C to pellet mitochondria. The pellet was washed in isolation buffer. Protein concentration was determined by the bicinchoninic acid protein assay (Thermo
Fisher Scientific). Samples were subjected to SDS-PAGE followed by immuno­
blot analysis. The intensities of KIF5 and dynein IC74 were normalized to
the mitochondrial marker Tom20. Relative purity of the mitochondrial fraction was assessed by sequentially immunoblotting the same membrane
with mitochondrial marker TOM20 and other organelle markers, including
EEA1 (endosomes), Grp78 (ER), and actin.
Statistical analysis
Images are representative of >10 cells as indicated from each figure legend from at least three repeats. All immunoblots are representative of at
least three experiments. For significance evaluation, two-tailed Student’s
t test was performed for normal distributed datasets. If the dataset did not
follow normal distribution, nonparametric Mann–Whitney U test was performed for two groups by Prism 5 (GraphPad Software). Measurements are
presented as means ± SEM.
Online supplemental material
Fig. S1 shows that SNPH selectively immobilizes axonal mitochondria.
Fig. S2 shows axonal mitochondria targeting of Miro1/Trak2. Fig. S3
shows that there is no binding between SNPH and DHC. Fig. S4 shows
a characterization of the SNPH–KIF5 interaction. Video 1 shows activity-dependent immobilization of axonal mitochondria, but not late endosomes, in snph+/+ neurons. Video 2 shows that deleting snph abolishes
activity-dependent immobilization of axonal mitochondria. Video 3 shows
that overexpressing SNPH selectively immobilizes axonal mitochondria,
but not late endosome, along the same axon. Video 4 shows immobilization of axonal mitochondria by applying field stimulation to wild-type
neurons. Video 5 shows that deleting snph abolishes field stimulation–
induced immobilization of axonal mitochondria. Video 6 shows that
expressing Trak2 increases axonal mitochondrial transport. Video 7 shows
that SNPH competes with Trak2 and abolishes axonal mitochondrial
motility. Video 8 shows that disrupting the KIF5–SNPH interaction reduces activity-dependent immobilization of axonal mitochondria. Video 9
shows that Ca2+ depletion enhances the axonal mitochondrial motility.
We thank Q. Cai and other members of the Sheng laboratory for helpful
discussions; M. Davis for editing; T.L. Schwarz, J.T. Kittler, P. Aspenström,
M. Peckham, F.A. Stephenson, R.J. Youle, and R.J. Diefenbach for DNA constructs and reagents; and the National Institute of Neurological Disorders and
Stroke (NINDS) Facility for DNA Sequencing and Protein Sequencing.
This work was supported by the Intramural Research Program of NINDS,
National Institutes of Health (Z.-H. Sheng) and an NINDS Competitive Fellowship (Y. Chen).
Submitted: 7 February 2013
Accepted: 10 June 2013
References
Banker, G.A., and W.M. Cowan. 1979. Further observations on hippocampal
neurons in dispersed cell culture. J. Comp. Neurol. 187:469–493. http://
dx.doi.org/10.1002/cne.901870302
Brickley, K., and F.A. Stephenson. 2011. Trafficking kinesin protein (TRAK)mediated transport of mitochondria in axons of hippocampal neurons.
J. Biol. Chem. 286:18079–18092. http://dx.doi.org/10.1074/jbc.M111
.236018
Cai, Q., and Z.H. Sheng. 2009. Moving or stopping mitochondria: Miro as a
traffic cop by sensing calcium. Neuron. 61:493–496. http://dx.doi.org/
10.1016/j.neuron.2009.02.003
Cai, Q., L. Lu, J.H. Tian, Y.B. Zhu, H. Qiao, and Z.-H. Sheng. 2010. Snapinregulated late endosomal transport is critical for efficient autophagylysosomal function in neurons. Neuron. 68:73–86. http://dx.doi.org/
10.1016/j.neuron.2010.09.022
Cai, Q., H.M. Zakaria, A. Simone, and Z.-H. Sheng. 2012. Spatial parkin translocation and degradation of damaged mitochondria via mitophagy in
live cortical neurons. Curr. Biol. 22:545–552. http://dx.doi.org/10.1016/
j.cub.2012.02.005
Carter, N., and R. Cross. 2001. An improved microscope for bead and surfacebased motility assays. Methods Mol. Biol. 164:73–89.
Chang, D.T., A.S. Honick, and I.J. Reynolds. 2006. Mitochondrial trafficking
to synapses in cultured primary cortical neurons. J. Neurosci. 26:7035–
7045. http://dx.doi.org/10.1523/JNEUROSCI.1012-06.2006
Chen, H., and D.C. Chan. 2009. Mitochondrial dynamics—fusion, fission,
movement, and mitophagy—in neurodegenerative diseases. Hum. Mol.
Genet. 18(R2):R169–R176. http://dx.doi.org/10.1093/hmg/ddp326
Chen, Y.M., C. Gerwin, and Z.H. Sheng. 2009. Dynein light chain LC8 regulates syntaphilin-mediated mitochondrial docking in axons. J. Neurosci.
29:9429–9438. http://dx.doi.org/10.1523/JNEUROSCI.1472-09.2009
Cho, K.I., H. Yi, R. Desai, A.R. Hand, A.L. Haas, and P.A. Ferreira. 2009.
RANBP2 is an allosteric activator of the conventional kinesin-1 motor
protein, KIF5B, in a minimal cell-free system. EMBO Rep. 10:480–486.
http://dx.doi.org/10.1038/embor.2009.29
Dunn, S., E.E. Morrison, T.B. Liverpool, C. Molina-París, R.A. Cross, M.C.
Alonso, and M. Peckham. 2008. Differential trafficking of Kif5c on
tyrosinated and detyrosinated microtubules in live cells. J. Cell Sci.
121:1085–1095. http://dx.doi.org/10.1242/jcs.026492
Encalada, S.E., L. Szpankowski, C.H. Xia, and L.S. Goldstein. 2011. Stable
kinesin and dynein assemblies drive the axonal transport of mammalian prion protein vesicles. Cell. 144:551–565. http://dx.doi.org/10
.1016/j.cell.2011.01.021
Fransson, S., A. Ruusala, and P. Aspenström. 2006. The atypical Rho GTPases
Miro-1 and Miro-2 have essential roles in mitochondrial trafficking.
Biochem. Biophys. Res. Commun. 344:500–510. http://dx.doi.org/10.1016/
j.bbrc.2006.03.163
Frezza, C., S. Cipolat, and L. Scorrano. 2007. Organelle isolation: functional
mitochondria from mouse liver, muscle and cultured fibroblasts. Nat.
Protoc. 2:287–295. http://dx.doi.org/10.1038/nprot.2006.478
Gross, S.P., M. Vershinin, and G.T. Shubeita. 2007. Cargo transport: two motors
are sometimes better than one. Curr. Biol. 17:R478–R486. http://dx.doi
.org/10.1016/j.cub.2007.04.025
Guo, X., G.T. Macleod, A. Wellington, F. Hu, S. Panchumarthi, M. Schoenfield,
L. Marin, M.P. Charlton, H.L. Atwood, and K.E. Zinsmaier. 2005.
The GTPase dMiro is required for axonal transport of mitochondria to
Drosophila synapses. Neuron. 47:379–393. http://dx.doi.org/10.1016/j
.neuron.2005.06.027
Hirokawa, N., R. Sato-Yoshitake, T. Yoshida, and T. Kawashima. 1990. Brain
dynein (MAP1C) localizes on both anterogradely and retrogradely transported membranous organelles in vivo. J. Cell Biol. 111:1027–1037.
http://dx.doi.org/10.1083/jcb.111.3.1027
Regulation of axonal mitochondrial transport • Chen and Sheng
Downloaded from on June 12, 2017
Kinesin ATPase activity assay and motor velocity measurement
Flag-KIF5C, Flag-SNPH and its truncated mutants, and Flag-Trak2 were expressed in HEK293 cells and then purified using anti-Flag M2 magnetic
beads (Sigma-Aldrich). The ATPase activity of KIF5C motor was measured
by the Kinesin ATPase Endpoint Assay Biochem kit (Cytoskeleton). In brief,
0.2 µg of purified Flag-KIF5C was incubated in the presence or absence of
0.5 µg Flag-SNPH or Flag-Trak2 in 30 µl of reaction buffer (15 mM Pipes,
5 mM MgCl2, 20 µM Taxol, and 20 µM acetyl–coenzyme A) for 30 min at
room temperature. After adding 2 µg MTs and 0.3 mM ATP to initiate the
reaction, the reaction continued for 5 min before termination by adding
70 µl CytoPhos for 10 min. The OD650 was measured by absorbance
microplate reader (SpectraMax 190; Molecular Devices). For KIF5 velocity
measurement, COS cells were transfected with EGFP-KIF5C and mCherry–
-tubulin or coexpressing with SNPH or Trak2 for 24 h. Time-lapse imaging
was performed using a confocal microscope (LSM 510 META) with a
C-Apochromat 63×, 1.4 NA oil immersion lens in modified Tyrode’s solution (10 mM Hepes, 10 mM glucose, 3 mM KCl, 145 mM NaCl, 1.2 mM
CaCl2, 1.2 mM MgCl2, and 1 µM glycine, pH 7.4). Time-lapse images
were collected at 256 × 256–pixel resolution (8 bit) with a 2.5-s interval.
The software RETRAC (Carter and Cross, 2001) was used to measure the
velocity of EGFP-KIF5C by tracking the EGFP-KIF5C puncta along MTs labeled with mCherry–-tubulin.
Online supplemental material is available at http://www.jcb.org/cgi/
content/full/jcb.201302040/DC1.
363
Published July 15, 2013
364
JCB • VOLUME 202 • NUMBER 2 • 2013
Tang, Y., and R.S. Zucker. 1997. Mitochondrial involvement in post-tetanic
potentiation of synaptic transmission. Neuron. 18:483–491. http://dx.doi
.org/10.1016/S0896-6273(00)81248-9
Vale, R.D. 2003. The molecular motor toolbox for intracellular transport. Cell.
112:467–480. http://dx.doi.org/10.1016/S0092-8674(03)00111-9
Verstreken, P., C.V. Ly, K.J. Venken, T.W. Koh, Y. Zhou, and H.J. Bellen.
2005. Synaptic mitochondria are critical for mobilization of reserve pool
vesicles at Drosophila neuromuscular junctions. Neuron. 47:365–378.
http://dx.doi.org/10.1016/j.neuron.2005.06.018
Wang, X., and T.L. Schwarz. 2009. The mechanism of Ca2+-dependent regulation of kinesin-mediated mitochondrial motility. Cell. 136:163–174.
http://dx.doi.org/10.1016/j.cell.2008.11.046
Yen, H.C., and S.J. Elledge. 2008. Identification of SCF ubiquitin ligase substrates by global protein stability profiling. Science. 322:923–929. http://
dx.doi.org/10.1126/science.1160462
Yi, M., D. Weaver, and G. Hajnóczky. 2004. Control of mitochondrial motility
and distribution by the calcium signal: a homeostatic circuit. J. Cell Biol.
167:661–672. http://dx.doi.org/10.1083/jcb.200406038\
Downloaded from on June 12, 2017
Hirokawa, N., S. Niwa, and Y. Tanaka. 2010. Molecular motors in neurons: transport mechanisms and roles in brain function, development, and disease.
Neuron. 68:610–638. http://dx.doi.org/10.1016/j.neuron.2010.09.039
Hollenbeck, P.J., and W.M. Saxton. 2005. The axonal transport of mitochondria.
J. Cell Sci. 118:5411–5419. http://dx.doi.org/10.1242/jcs.02745
Ilangovan, U., W. Ding, Y. Zhong, C.L. Wilson, J.C. Groppe, J.T. Trbovich, J.
Zúñiga, B. Demeler, Q. Tang, G. Gao, et al. 2005. Structure and dynamics
of the homodimeric dynein light chain km23. J. Mol. Biol. 352:338–354.
http://dx.doi.org/10.1016/j.jmb.2005.07.002
Jiang, M., and G. Chen. 2006. High Ca2+-phosphate transfection efficiency
in low-density neuronal cultures. Nat. Protoc. 1:695–700. http://dx.doi
.org/10.1038/nprot.2006.86
Kang, J.S., J.H. Tian, P.Y. Pan, P. Zald, C. Li, C. Deng, and Z.-H. Sheng. 2008.
Docking of axonal mitochondria by syntaphilin controls their mobility and affects short-term facilitation. Cell. 132:137–148. http://dx.doi
.org/10.1016/j.cell.2007.11.024
Lao, G., V. Scheuss, C.M. Gerwin, Q. Su, S. Mochida, J. Rettig, and Z.-H.
Sheng. 2000. Syntaphilin: a syntaxin-1 clamp that controls SNARE
assembly. Neuron. 25:191–201. http://dx.doi.org/10.1016/S0896-6273
(00)80882-X
Ligon, L.A., M. Tokito, J.M. Finklestein, F.E. Grossman, and E.L. Holzbaur.
2004. A direct interaction between cytoplasmic dynein and kinesin I may
coordinate motor activity. J. Biol. Chem. 279:19201–19208. http://dx.doi
.org/10.1074/jbc.M313472200
Ma, H., Q. Cai, W. Lu, Z.H. Sheng, and S. Mochida. 2009. KIF5B motor adaptor syntabulin maintains synaptic transmission in sympathetic neurons.
J. Neurosci. 29:13019–13029. http://dx.doi.org/10.1523/JNEUROSCI
.2517-09.2009
MacAskill, A.F., and J.T. Kittler. 2010. Control of mitochondrial transport
and localization in neurons. Trends Cell Biol. 20:102–112. http://dx.doi
.org/10.1016/j.tcb.2009.11.002
MacAskill, A.F., K. Brickley, F.A. Stephenson, and J.T. Kittler. 2009a. GTPase
dependent recruitment of Grif-1 by Miro1 regulates mitochondrial trafficking in hippocampal neurons. Mol. Cell. Neurosci. 40:301–312. http://
dx.doi.org/10.1016/j.mcn.2008.10.016
MacAskill, A.F., J.E. Rinholm, A.E. Twelvetrees, I.L. Arancibia-Carcamo, J.
Muir, A. Fransson, P. Aspenstrom, D. Attwell, and J.T. Kittler. 2009b.
Miro1 is a calcium sensor for glutamate receptor-dependent localization of mitochondria at synapses. Neuron. 61:541–555. http://dx.doi
.org/10.1016/j.neuron.2009.01.030
Miller, K.E., and M.P. Sheetz. 2004. Axonal mitochondrial transport and potential are correlated. J. Cell Sci. 117:2791–2804. http://dx.doi.org/10.1242/
jcs.01130
Misko, A., S. Jiang, I. Wegorzewska, J. Milbrandt, and R.H. Baloh. 2010.
Mitofusin 2 is necessary for transport of axonal mitochondria and interacts with the Miro/Milton complex. J. Neurosci. 30:4232–4240. http://
dx.doi.org/10.1523/JNEUROSCI.6248-09.2010
Pilling, A.D., D. Horiuchi, C.M. Lively, and W.M. Saxton. 2006. Kinesin-1
and Dynein are the primary motors for fast transport of mitochondria in
Drosophila motor axons. Mol. Biol. Cell. 17:2057–2068. http://dx.doi
.org/10.1091/mbc.E05-06-0526
Popov, V., N.I. Medvedev, H.A. Davies, and M.G. Stewart. 2005. Mitochondria
form a filamentous reticular network in hippocampal dendrites but are
present as discrete bodies in axons: a three-dimensional ultrastructural
study. J. Comp. Neurol. 492:50–65. http://dx.doi.org/10.1002/cne.20682
Rintoul, G.L., A.J. Filiano, J.B. Brocard, G.J. Kress, and I.J. Reynolds. 2003.
Glutamate decreases mitochondrial size and movement in primary forebrain neurons. J. Neurosci. 23:7881–7888.
Russo, G.J., K. Louie, A. Wellington, G.T. Macleod, F. Hu, S. Panchumarthi, and
K.E. Zinsmaier. 2009. Drosophila Miro is required for both anterograde
and retrograde axonal mitochondrial transport. J. Neurosci. 29:5443–
5455. http://dx.doi.org/10.1523/JNEUROSCI.5417-08.2009
Saotome, M., D. Safiulina, G. Szabadkai, S. Das, A. Fransson, P. Aspenstrom, R.
Rizzuto, and G. Hajnóczky. 2008. Bidirectional Ca2+-dependent control
of mitochondrial dynamics by the Miro GTPase. Proc. Natl. Acad. Sci.
USA. 105:20728–20733. http://dx.doi.org/10.1073/pnas.0808953105
Sheng, Z.H., and Q. Cai. 2012. Mitochondrial transport in neurons: impact on
synaptic homeostasis and neurodegeneration. Nat. Rev. Neurosci. 13:77–
93. http://dx.doi.org/10.1038/nrn3156
Smith, M.J., K. Pozo, K. Brickley, and F.A. Stephenson. 2006. Mapping the
GRIF-1 binding domain of the kinesin, KIF5C, substantiates a role
for GRIF-1 as an adaptor protein in the anterograde trafficking of cargoes. J. Biol. Chem. 281:27216–27228. http://dx.doi.org/10.1074/jbc
.M600522200
Stowers, R.S., L.J. Megeath, J. Górska-Andrzejak, I.A. Meinertzhagen, and
T.L. Schwarz. 2002. Axonal transport of mitochondria to synapses depends on milton, a novel Drosophila protein. Neuron. 36:1063–1077.
http://dx.doi.org/10.1016/S0896-6273(02)01094-2